1. Field of Invention
This invention relates to a method for detecting an antigen in a biological sample suspected of containing the antigen. The invention has particular application to detecting gram-negative bacteria in a clinical specimen.
Numerous techniques are known for detecting antigens in a sample, such as a biological fluid, i.e., blood, urine, cell cultures. It is important to be able to quickly and accurately detect the presence of antigens such as gram-negative bacteria, i.e., Chlamydia trachomatis, Chlamydia psittaci and Neisseria gonorrhoeae because of the prevalence of diseases associated with such bacteria. Many of the techniques utilized to detect antigens involve cell culture procedures. Such procedures are relatively long and complicated and the results are greatly dependent on the skill of the technician. Many other techniques, for example electrophoresis, require complicated and/or costly instruments that are not readily available.
Many of the detection techniques involve immunoassays. Some of these techniques involve detecting antibodies to the antigen of interest. Such indirect techniques tend to be inaccurate because antibodies often remain in the human body after the disease has been cured. Therefore, it is preferable to assay for antigens rather than antibodies. Immunoassay techniques for the detection of antigens often involve enzyme immunoassays such as the enzyme linked immunosorbent assay generally referred to as ELISA. Such assays typically involve detecting the antigen of interest by coating the antigen on a bare solid surface or a surface that has been pre-coated with a protein such as an antibody. After coating the surface of the support with the antigen, the surface is washed to remove unbound antigen. Thereafter, the antigen on the surface is contacted with antibody for the antigen that is labeled or is capable of being labeled. The surface is again washed and the antibody that has bound to the surface of the support is detected by detecting the label.
The number of washing steps required in the above procedure adds significant time and manipulation to the assay. There is, therefore, a need for a method for detecting antigens, especially antigens from gram-negative bacteria, that is more rapid, reliable and cost effective than tests now known or available. It is likewise important that such a test require less manipulation than presently available tests.
2. Description of Related Art
A method for determining Chlamydia trachomatis antigen in a clinical specimen is disclosed in U.S. Pat. No. 4,497,899. The method disclosed involves lysing Chlamydia cells in the specimen to release the antigen; coating a bare solid support with the cell lysate; separating the coated support from the specimen; treating the separated support with antibody to form an antigen-chlamydia antibody complex on the support; separating the complex from unbound antibody; treating the bound complex with labeled antiglobulin to form an antigen-antibody-labeled antiglobulin complex on the support; separating the latter complex from unbound labeled antiglobulin; and determining bound labeled antiglobulin as a measure of antigen in the specimen. U.S. Pat. No. 4,497,900 discloses a method for determining Neisseria gonorrhoeae analogous to that in U.S. Pat. No. 4,497,899.
An indirect method for determining an antigen in a liquid sample is disclosed in U.S. Pat. No. 4,067,959 involving adsorbing antigen of the same immunological type as the sample antigen onto a solid support surface, reacting a known quantity of specific labeled antibody in solution with the sample antigen and with the adsorbed antigen. The labeled antibody is in excess so that a portion of the labeled antibody is bound in the solution to the sample antigen and excess labeled antibody immunologically reacts with the adsorbed antigen on the surface. The surface is washed and then the quantity of reacted labeled antibody on the surface is determined as a measure of the antigen in the sample.
A method for assaying for the presence of a Chlamydial infection involving generating antibodies against the principal outer membrane protein of Chlamydia trachomatis is disclosed in U.S. Pat. No. 4,427,782. The method comprises treating a sample suspected of containing chlamydial infection to solubilize the Chlamydia outer cell membrane protein, contacting the generated antibodies with the solubilized specimen and determining the reaction between the antibodies and the solubilized specimen. European Patent Application 0,174,106 discloses a method of detection of cell membrane protein, especially principal outer membrane of Chlamydia trachomatis. Great Britain Patent Application Ser. No. 2 047 889A discloses serological testing for Chlamydia trachomatis antibodies by microimmunofluorescence of utilizing a formaldehyde stabilized antigen.
U.S. Pat. Nos. 4,145,406 and 4,254,082 disclose a method for determining a specific binding substance in a liquid sample employing a nonspecific adsorbent and a labeled form of a specific binding partner to the substance to be determined. U.S. Pat. No. 4,188,371 discloses a method employing immunoradiometric assay techniques for detecting Neisseria bacteria in a sample. The method involves lysing bacteria in the sample to be tested; contacting the lysate with radiolabeled antisera; incubating the resultant mixture and then contacting the mixture with a solid phase antigen or an antigen immunoadsorbent; incubating the resultant mixture and then monitoring the radioactivity of the supernatant.